3 RESULTS AND DISCUSSION
In 2021, TZJ2134 was collected in Shandong Province in China and showed
positivity for PRRSV-1 by detection using primer L12 (Table S1).
Subsequently, a sequence of TZJ2134 (TZJ2134-L12) was obtained by
amplification with the detection primer L12, located in the partial Nsp2
gene (nt 1672-nt 2112 of DV) (Figure 1C). Phylogenetic analysis showed
that all Chinese PRRSV-1 isolates belonged to subtype 1 and could be
divided into four subgroups (Amervac-like, BJEU06-1-like, HKEU16-like,
and NMEU09-1-like isolates) (Figure 1). TZJ2134-L12 belonged to DV-like
isolates and shared the highest sequence identity (99.54%) with the DV
vaccine strain (Table 1).
To obtain the complete genome of TZJ2134, we designed eight pairs of
primers for amplification. Unfortunately, probably due to the low viral
load, the whole-genome sequence could not be obtained after repeated
attempts, and only two overlapping fragments of TZJ2134 were amplified
by using the primers Ly-E and Ly-F (Table S2). Subsequently, the
resulting sequences of two overlapping fragments were assembled into a
contig (named TZJ2134-(A+B)). Further
genetic evolution and homology
analyses showed that TZJ2134-(A+B) located
at nt 7463-nt 11272 (3810 nt in
length) in partial Nsp9, complete Nsp10 and partial Nsp11 (Figure 1C)
shared 97.1% nucleotide identity with the DV vaccine strain and 97.4%
nucleotide identity with the Amervac vaccine strain (Table 1). To
establish a genetic relationship between TZJ2134-(A+B) and other PRRSV-1
isolates, we constructed a
phylogenetic tree based
on 65 PRRSV-1 strains (Table S1).
Phylogenetic analysis showed that TZJ2134-(A+B) was intermediate between
Amervac-like isolates and DV-like isolates and formed a separate
subgroup (DV+Amervac-like isolates) with PRRS-FR-2014-56-11-1,
DK-2011-05-23-9 and OLot/91 strains (Figure 1B).
RDP4 and SimPlot (version 3.5.1) were used to test for recombination of
TZJ2134-(A+B). The RDP4 analysis results showed that TZJ2134-(A+B) was a
recombinant strain from Amervac and DV vaccine strains with a potential
crossover event spanning Nsp10. Additionally, the recombination event
was further confirmed by SimPlot 3.5.1, which showed that the
recombination breakpoint was approximately located in Nsp10 (nt 9423)
(Figure 2A). Based on the putative recombination breakpoint (nt 9243),
we divided TZJ2134-(A+B) into two fragments, TZJ2134-A (nt 7463-nt 9423)
and TZJ2134-B (nt 9423-nt 11272), for phylogenetic and homology
analyses. The results revealed that the homology between the two
fragments and the corresponding
parent viruses showed high similarity (Table 1). TZJ2134-A shared the
highest nucleotide identity (99.17%) with the DV vaccine strain (Table
1) and belonged to DV-like isolates (Figure 2B). TZJ2134-B shared the
highest nucleotide identity (99.73%) with the Amervac vaccine strain
(Table 1) and belonged to Amervac-like isolates (Figure 2C). Both the DV
and Amervac vaccine strains were PRRSV-1 MLV strains.
To the best of our knowledge,
only two reports, from France and Denmark, have described recombination
events between two PRRSV-1 MLV strains (Kvisgaard et al., 2020; Renson
et al., 2017). One of them, PRRS-FR-2014-56-11-1, was the first
recombinant strain derived from the Amervac vaccine strain and the DV
vaccine strain described previously, with recombination events occurring
at nt 500 to nt 1370, nt 3646 to nt 4272 and nt 4972 to nt 8430 in ORF1,
as determined using RDP4 (Renson et al., 2017). Homology analysis showed
that TZJ2134-(A+B) has the highest nucleotide identity (97.6%) with
PRRS-FR-2014-56-11-1.
PRRS-FR-2014-56-11-1 and
TZJ2134-(A+B) are intermediates between Amervac-like isolates and
DV-like isolates with DK-2011-05-23-9 and OLot/91 strains in the
phylogenetic tree (Figure 1B). The recombinant and phylogenetic analysis
results showed that all three viruses were recombinant strains derived
from the Amervac vaccine strain and DV vaccine strain but with different
recombinant patterns (Figure S1) and formed a novel subgroup
(DV+Amervac-like isolates) in the phylogenetic tree (Figure 1B).
In the late 1990s, PRRSV-1 MLVs were usually used to control PRRSV-1
infection in Europe (Chae, 2021). PRRSV-1 MLVs used worldwide include
Porcilis PRRS (MSD), Amervac PRRS (Laboratories Hipra S.A.), ReproCyc
PRRS EU (Boehringer Ingelheim), Ingelvac PRRSFLEX EU (Boehringer
Ingelheim), Pyrsvac-183 (SYVA Laboratories), and Ingelvac
PRRSFLEX® EU (Boehringer Ingelheim) (Nan et al.,
2017). The Amervac vaccine strain is usually produced from the Amervac
PRRS vaccine introduced by Hipra, and the DV vaccine strain is usually
produced from the Porcilis® PRRS vaccine introduced by MSD. Both
vaccines are often used in western Europe. The present findings confirm
that animals infected with the recombinant strain showed a viremia level
10- to 100-fold higher in than that in animals infected with the Amervac
or DV vaccine strain, in both inoculated and contact pigs (Eclercy et
al., 2019). However, TZJ2134 may have had a low viral load, so the
complete genome sequence could not be obtained. This study provides the
first genetic evidence of the recombination of the Amervac vaccine
strain with the DV vaccine strain in China. As the largest pork importer
in the world, in China, the pig industry is vulnerable to the influence
of the foreign pig industry (Brockmeier et al., 2012; van Geelen et al.,
2018; H. L. Zhang et al., 2018). TZJ2134 may be highly likely to be
introduced from Europe via pig trade after recombination abroad,
as PRRSV-1 MLV is not currently
allowed for use in mainland China. Although recombination of MLV strains
is rarely reported, the existence of TZJ2134 is a reminder that
surveillance against PRRSV-1 should be strengthened in China.